IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity

During wound healing, fibroblasts are recruited from the surrounding tissue to accomplish repair. The requisite migration and proliferation of the fib roblasts is promoted by growth factors including those that activate the ep idermal growth factor receptor (EGFR), Counterstimulatory factors in wound fluid are postulated to limit this response; among these factors is the ELR -negative CXC chemokine, interferon inducible protein-10 (IP-10),We report here that IP-10 inhibited EGF- and heparin-binding EGF-like growth factor-i nduced Hs68 human dermal fibroblast motility in a dose-dependent manner (to 52% and 44%, respectively, at 50 ng/ml IP-10), whereas IP-10 had no effect on either basal or EGFR-mediated mitogenesis (96 +/- 15% at 50 ng/ml). The se data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility. To define the molecular basis of this negative transmodulation of EGFR sign aling, we found that IP-10 did not adversely impact receptor or immediate p ostreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effecters phospholipase C-gamma and erk mitogen-activa ted protein kinases, Morphological studies suggested which biophysical step s may be affected by demonstrating that IP-10 treatment resulted in an elon gated cell morphology reminisce:nt of failure to detach the uropod; in supp ort of this, IP-10 pretreatment inhibited EGF-induced cell detachment. Thes e data suggested that calpain activity may be involved. The cell permeant a gent, calpain inhibitor I, limited EGF-induced motility and de-adhesion sim ilarly to IP-10, IP-10 also prevented EGF induced calpain activation (reduc ed by 71 +/- 7%).That this inhibition of EGF-induced calpain activity was s econdary to IP-10 initiating a cAMP-protein kinase A-calpain cascade is sup ported by the following evidence: (a:) the cell permeant analogue 8-(4-chlo rophenylthio)-cAMP (CPT-cAMP) prevented EGF-induced calpain activity and mo tility; (b) other ELR-negative CXC chemokines, monokine induced by IFN-gamm a and platelet factor 4 that also generate cAMP, inhibited EGF-induced cell migration and calpain activation; and (c) the protein kinase A inhibitor R p-8-Br-cANIPS abrogated IP-10 inhibition of cell migration, cell detachment , and calpain activation. Our findings provide a model by which IP-10 suppr esses EGF-induced cell motility by inhibiting EGF-induced detachment of the trailing edges of motile cells.